The objective of the proposed research is to define in molecular terms the interactions between the vitamin K-dependent blood coagulation factors (in this context, II, VII, IX, and X), calcium ions, and phospholipid. A model system consisting of bovine prothrombin fragment-1 (residues 1-156) will be employed. Correlations will be sought between fragment-1 structure and its functional properties, namely metal ion binding and metal ion-dependent phospholipid binding. We plan to explore the importance of a variety of structural features of fragment-1 to the function of fragment-1 as reflected by such physical properties as: optical spectroscopic, metal ion binding, and the ability of the protein to self-associate and/or to associate with phospholipid. We will examine ionization behavior via potentiometry, fluorescence spectroscopy, and calcium and magnesium nmr. The observed prototropic equilibria will be correlated with the primary structure of fragment-1 and the ability of the protein to bind metal ions and phospholipid. The importance of side-chain interactions to metal ion binding and metal ion-dependent phospholipid binding will be studied by selective chemical modification of various side-chain functional groups. We also intend to carry out studies designed to deduce the structure of the metal complexes formed with fragment-1. These studies will involve metal ion nmr and metal ion fluorescence techniques.